SUMMARY RNA polymerase III (Pol III) is increasingly recognized as an important drug target and a rapidly developing area of academic research. Pol III is responsible for generating RNAs that are critical for cell growth, such as tRNAs, certain rRNAs, and various noncoding RNAs involved in processes such as mRNA splicing. These RNAs are required for cell growth, proliferation, and differentiation and disrupting the pathways that control Pol III is now thought to be valuable for targeting cancer and obesity. Despite the importance of Pol III, little is known about the pathways that regulate Pol III transcription. This is largely due to the absence of simple and robust reporters for Pol III activity. This is in contrast to reporter assays for Pol II promoters for mRNA transcription?these reporters comprise Pol II promoters controlling reporter genes such as green fluorescent protein, and have been the major driver for drug discovery for mRNA transcription inhibitors. However, Pol III generates RNAs that cannot be translated. As a result, nearly all studies of Pol III rely on Northern blotting, which is slow and not suited to high-throughput screening efforts. At Lucerna, we have developed a photostable RNA mimic of red fluorescent protein (termed Corn) and demonstrated that this RNA can serve as a robust fluorescent reporter for Pol III promoters. By simply creating a Pol III promoter that controls the synthesis of the Corn RNA, fluorescence can be readily detected without the need to synthesize a protein. We have demonstrated this concept using the 5S subtype of a Pol III promoter. Interestingly, we found marked cell-to-cell variation in Pol III transcription after cells are exposed to serum and other growth-promoting signals. Therefore, the goal of this Phase I SBIR project is to develop this simple and genetically encodable Corn reporters for live cell imaging and measurement of all three subtypes of Pol III transcription. Further, we will develop this reporter system so that it can be used in flow cytometry analysis, allowing Pol III transcription to be monitored in hundreds of thousands of cells over time. For phase II, we will adapt our Pol III reporters into marketable toolkits, validate the Pol III reporters in disease-relevant cell lines, and use the Cas9/CRISPR technology to image endogenous Pol III transcription activity. Together, the plasmids and kits generated in this project will result in the first Pol III reporters, and will therefore constitute an important commercial product that will have wide utility in the academic RNA research community and will enable discovery of small molecule inhibitors of this previously hard-to-assay class of drug targets.